Vol. 10(10), pp. 1031-1042, 5 March, 2015 DOI: 10.5897/AJAR2014.9305 Article Number: 672CD2751172 ISSN 1991-637X Copyright ©2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR
African Journal of Agricultural Research
Full Length Research Paper
An efficient protocol for indirect somatic embryogenesis and shoot organogenesis from leaf segments of date palm (Phoenix dactylifera L.) CV. Quntar Ahmed Madi Waheed AL-Mayahi Date Palm Research Centre, Basra University, Basra, Iraq. Received 4 November, 2014; Accepted 21 February, 2015
The establishment of an efficient protocol for plantlets regeneration and micropropagation from leaf cultures of date palm cv. Quntar is reported. The two types of leaflet segments used in the experiment (First piece of leaf base which have white color, and second piece of leaf tip which have green color), were taken from micro propagated shoots of date palm cv. These leaf segments were in vitro culture on MS media containing various concentrations of 2,4-dichlorophenoxyaceticacid at 1.0 mgl-1 and benzyl adenine (BA). Maximum induction of callus was obtained from piece of leaf base (white) in medium supplemented with 5.0 mgl-1 2,4-D and 1.0 mgl-1 (BA), as compare with [piece of leaf tip (green)] which did not show response for callus formation. Using culture system with temporary tissue immersions "bioreactor" in Somatic embryo production enriched with 1.0 mgl-1 2,4-D + 500 mgl-1 active charcoal (AC) gave the highest number of somatic embryos (172 embryos). These embryos formed from 500 mg initial callus. As well as, the short immersions frequently repeated, 1 min immersions every 4 h, led to the largest quantities of embryos. Indirect shoot organogenesis was achieved from the embryogenic callus using 3.0 mgl-1 2iP + 1.0 mgl-1 NAA, where highest number of shoots (8.28 ± 0.71) with maximum frequency (58.34%) was regenerated. The addition of 3 gml-1 Gelrite to medium was optimal for the shoots proliferation. The balance of auxin to cytokinin in medium affected on the growth of the date palm tissue. The shoots were successfully rooted (80%) with rapid elongation when cultured on MS medium containing 0.5 mgl-1 GA3 + 1.0 mgl-1 NAA. Rooted shoots were successfully acclimatized when planted in plastic pots containing a mixture of peat moss and perlite (2:1) with 80% success. Key words: Date palm, histology, indirect shoot organogenesis, leaf segment, somatic embryogenesis, temporary immersion ‘bioreactor’.
INTRODUCTION Date palm (Phoenix dactylifera L.) cultivation is one of the most economically-important activities in the arid zones
of the Middle East and North Africa (El Hadrami and AlKhayri, 2012). One of the major problems in date palm
E-mail:
[email protected] Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License
1032
Afr. J. Agric. Res.
cultivation that prevents rapid crop improvement is the lack of an adequate method of asexual propagation. Traditionally, date palm is propagated vegetatively by offshoots produced by desirable trees. The low rate of propagation (1-20 offshoots/ tree, depending on the variety) has limited the multiplication of healthy plants (Tisserat, 1982). To overcome the propagation problems and to maintain the germplasm, the in vitro micropropagation. Large-scale plant production through cell tissue and embryo cultures using bioreactors is promising for industrial plant propagation. Bioreactors are usually described in a biochemical context as selfcontained, sterile environments which capitalize on liquid nutrient or liquid/air inflow and outflow systems, designed for intensive culture and according maximal opportunity for monitoring and control over micro environmental conditions such as agitation, aeration, temperature, dissolved oxygen and pH (Paek et al., 2005). The application of liquid cultures for micropropagation in bioreactors using the embryogenic and organogenic regeneration pathways is becoming a more efficient alternative system for scale-up and automation in vitro (Ziv, 1995; Tahardi et al., 2003; Ducos et al., 2007). However, somatic embryogenesis appears to be the most appropriate for an automated system in liquid medium (Etienne et al., 2006). The cultivation in liquid media using a temporary immersion system with different frequencies of immersion was reported to improve plant quality and multiplication rates of banana and coffee (Alvard et al., 1993; Teisson and Alvard, 1995). In addition, Othmani et al. (2009) demonstrated the applicability of embryogenic suspension culture for high output of date palm somatic embryos using temporary immersion bioreactor (TIB) system. The objective of the present study is to develop the use of other possible explants (leaf segments) as the sources of in vitro culture for date palm cv. Quntar, as well as development of somatic embryos in culture system with temporary tissue immersions "bioreactor", and optimize the protocol for indirect shoot.
27±2°C in darkness. The frequency of explant producing callus and fresh weights (gm) of callus were recorded for callusing explants after 6 weeks. The primary calluses were chopped and transferred on the same medium, after two subcultures with six weeks interval, embryogenic calluses were used for the installation of embryogenic suspension cultures and indirect shoot organogenesis .
PLANT DEVELOPMENTAL PATHWAYS FROM EMBRYOGENIG CALLUS Somatic embryogenesis Effect of culture media composition in culture system with temporary tissue immersions "bioreactor" on Somatic embryo production Embryogenic callus pieces (500 mg), [developed from piece of leaf base which has white color of date palm (cv. Quntar)] were culturing in bioreactor with 400 ml liquid medium (Figure 2A). The culture medium contained MS basal salts, 170 mgl-1 KH2PO4, 10 mgl-1 thiamine-HCl, 200 mgl-1 glutamine, 30 gl-1 sucrose and 0.500 gl-1 AC. This medium supplemented with different concentrations of 2,4D (0.0, 0.5, 1.0 and 2.0 mgl-1). A temporary immersion system (TIS) was conducted using bioreactor. The callus in bioreactors remained in their culture containers at 5 weeks. The fresh mass of callus was evaluated at every subculture. The immersion duration for the latter was 3 min every 8 h. Once the best combination of culture medium was determined, a second experiment was carried out, to evaluate the effect of the immersion period on somatic embryo development as follows: 1 min every 4 h, 5 min every 8 h and 10 min every 12 h (Figure 2B). In both two experiments, three bioreactors containers were used (Figure 2C). The suspension cultures were kept under 16 h light photoperiod (30 µmol m-2 s-1) at 27 ± 2°C. For both experiments, the total number of somatic embryos, was determined after 45 days from culture.
Germination of the somatic embryos Matured somatic embryos of date palm were transferred to MS medium enriched with 0.1 mg l-1 NAA. The MS basal medium was added with 30 g/L sucrose, pH 5.7 and solidified with 6 gl-1 Agar for shoot and root induction. The cultures were incubated in the culture room at 27 ± 2°C with 16 h light and 8 h dark.
Organogenic pathway MATERIALS AND METHODS
Indirect shoot organogenesis from leaf derived callus
Plant material
For shoot regeneration, embryonic callus obtained from the above experiments was transferred to MS medium supplemented with various of cytokinins (BA alone, 2iP alone or in combination with auxin NAA, for each experiment a minimum of 12 replicates. The data was recorded after 12 weeks from culturing as following: The percentage of response of callus mass on shoot regeneration; number of regeneration shoots /explant. For improvement of shoot regeneration, the medium was optimized by testing the effect of different concentrations of gelling agents (6, 7, and 8 gl-1 agar or 2, 3, and 4 gl-1 Gelrite). Cultures were maintained at 27 ± 2°C in a growth chamber with a 16 h photo-period under standard cool white fluorescent tubes (35 µmol s-1 m-2) and 60 to 70% relative humidity.
The experiment was carried out at the Plant Tissue Culture Laboratory, Basra University, Iraq. Two types of leaf segments (piece of leaf base which has white color, and piece of leaf tip which has green color) (Figure 1A and B). These pieces were taken from plantlets of date palm (P. dactylifera L. cv. Quntar) produced in in vitro culture. After, the larger cut of leaf was trimming into segments at 1 cm length. To induce callus, these leaf segments were horizontally cultured directly in Murashige and Skoog, 1962).’MS medium’, with additional 100 mgl-1 glutamine, 5 mgl-1thiamine HCl, 1 mgl-1 biotin, 40 mgl-1adenine sulfate, 30 gl-1 sucrose, 2.0 gl-1 activated charcoal. Also the media were supplemented with different concentrations of 2,4-D (0.0, 1.0, 2.5 and 5 mgl-1) combined with 1 mgl-1 BA. The pH of medium was adjusted to 5.8 before adding 7 gl-1 agar. The media were sterilised by autoclaving at 1.1 kg cm-2 (121°C) for 20 min. All the cultures were incubated at
Elongation and rooting Regeneration shoots were transferred to the MS medium
AL-Mayahi
1033
B. leaf tip
A. leaf base
B
A
Figure 1. Adventitious shoot induced from apical bud of date palm (P. dactylifera L. cv. Quntar)" Two types of leaf segments are used in this study": A) piece of leaf base which has white color. B) Piece of leaf tip which has green color.
B
A
Filters
C
Timer
Pump
Figure 2. (A) Date palm callus cv. Quntar cultured in bioreactor. (B) The timer organize the immersion periods through the pump in bioreactor. (2-C) Bioreactors that used for induction of somatic embryo from culture of embryonic callus.
supplemented with 0.5 mgl-1 GA3 + 1.0 mg1-1 NAA for elongation and rooting.
room (25±2°C, 55±5% RH, under 16 h of photoperiod with a light intensity of 40 μmol m-2 s-1) for 45 days. The glass bottles were gradually removed upon emergence of new leaves and acclimatized plantlets were transferred to the greenhouse.
Acclimatization All the produced plantlets from indirect regeneration via somatic embryogenesis and shoot organogenesis were acclimatized. The plantlets were gently removed from the vessels, washed initially to remove adhered gelling agent and traces of the medium to avoid contamination. Then, the plantlets were washed with distilled water and treated with fungicide (Benlet 500 mgl-1) for 20 min and transferred to plastic pots containing autoclaved a mixture of peat moss and perlite (2:1) (AL-Mayahi, 2014). The plants were covered with glass bottles to maintain humidity. The plants were initially irrigated with quarter-strength inorganic salts of MS medium for 2 week followed by tap water. Potted plantlets were grown in culture
Histological studies Histological examinations during bud formation were carried out using a freezing microtome. Microtome slide preparation and observation were made following the methods as described by Sarker and Awal (1999).
Experimental design and statistical analysis A complete randomized design was employed in all of the
1034
Afr. J. Agric. Res.
Figure 3. Callus induction from leaf segments of date palm cv. Quntar (A) Swelling of leaf explants. (B) Initial callus tissue in MS 5 mgl-1 2-4,D+1.0 mgl-1 BA (C). granular callus showing the embryogenic structures on same medium after two subcultures with six weeks interval from culture of primary callus.
experiments. Analysis of variance was used to show statistical differences between treatments using the L.S.D. at 5% (Snedecor and Cochran, 1989).
RESULTS Formation of primary and embryogenic callus The first evidence of date palm callus induction process was the swollen of leaf segment when it cultured on MS medium supplemented with different concentrations of 24,D+ BA after 3 weeks of incubation (Figure 3A). Maximum response callusing response from the [piece of leaf base (white)], reached 53.34% in the medium MS + 5 mgl-1 2-4,D+1.0 mgl-1 BA after 5 weeks of incubation (Figure 3B). These same media gave the highest fresh weight of callus reached 1.239 g. (Table 1). The weight in this treatment which was statistically significant compared with the other treatments media.. Also, variations existed between two types of leaves segments in their ability to form callus. The piece of leaf base produced considerably more callus than piece of leaf tip which did not show any response for callus formation (Table 1). When the globular and compact primary callus were cultivated on the same medium. This callus gave rise to friable granular callus composed of embryogenic cells after two subcultures with six weeks interval (Figure 3C). According to the results shown in Table 1, leaves cultured on MS medium supplemented with 5 mgl-1 24,D+1.0 mgl-1 BA gave better callus formation. The ratio of auxin: cytokinin was critical for inducing callus at a high frequency. Using certain hormonal balance in which the percentage of the auxins is much bigger than the percentage of the cytokenines, results in unbinding and breaking up the cohesive tissue structure of the basic explant, and also in cell division, which led to important
swelling in the size of the basic explants, followed by the appearance of the callus tissue. Of all the auxins or auxin-like plant growth regulators, 2,4-D has proven extremely useful, being used in 57.1% of successful embryogenic cultures (Al-Khalifah and Shanavaskhan, 2012). Callus induction has been reported from almost all types of tissues of date palm on various media formulations, which usually contained a combination of an auxin and a cytokinin (Taha et al., 2002; Al-Mayahi 2013). The type of tissue reactions were influenced by many factors but mostly by medium composition and the level of tissue differentiation and lignification (Abahmane, 2013). This good response of explants for callus formation may be due to the efficient selection of cells in an explants (Al-Mallah and Hassan, 1994). Coupled with compatibility between the endogenous hormonal levels and the addition of growth regulator to the induction medium. This may also suggest that levels of endogenous hormones or their sensitivity might vary between organs. These results are similar to those reported by Sane et al. (2012) who confirmed that the aptitude for primary callogenesis appeared to be strongly dependent on the explant nature, the growth regulators used and the genotype. Effect of culture media composition in culture system with temporary tissue immersions "bioreactor" on Somatic embryo regeneration The effect of the four liquid media supplemented with different concentrations of 2,4-D in culture system with temporary tissue immersions on somatic embryo formation from embryogenic callus derived from piece of leaf base (white) (Table (2). The highest number of somatic embryos was obtained in media containing 1.0 mgl-1 2,4-D After 45 days of culture reached 172
AL-Mayahi
1035
Table 1. Effect of different combinations of growth regulators on callus proliferation from two types of leaf segments of date palm c.v. Quntar.
Types of leaf segments
Piece of leaf base (white)
Piece of
leaf tip (green)
Treatments (mgl-1) 0.0 (1.0) 2-4,D+ BA(1.0) (2.5) 2-4,D+ BA(1.0) (5.0) 2-4,D+ BA(1.0)
Induction callus (%) 0.0 c 6.67 c 26.67 b 53.34 a
Fresh weight of callus (g) 0.0 d 0.466 c 1.017 b 1.239 a
0.0 (1.0) 2-4,D+ BA(1.0) (2.5) 2-4,D+ BA(1.0) (5.0) 2-4,D+ BA(1.0)
-
-
* ± Standard error (n = 15). *No response. ** Values followed by the same letter are not significantly different at P
